This project is to identify DNA sequences which are responsible for transcriptional stimulation of gene expression by prolactin and to isolate and clone the DNA binding proteins responsible for those actions. Prolactin is a member of a diverse family of regulatory proteins. Prolactin is primarily involved in pregnancy and lactation in human females and has been implicated in some breast and prostatic cancers. Other members of this family are important in growth regulation, metabolic regulation and development. The first aim of this project is to measure transcriptional regulation of chimeric promoter/reporter genes in cell cultures. The promoter will be derived from the prolactin-stimulated cp35 gene of the pigeon cropsac. This gene's structure and regulation have been extensively investigated and preliminary studies show that it contains sequences which specifically bind nuclear proteins. This promoter also supports in vitro transcription. Proteins extracted from nuclei of prolactin- stimulated tissues stimulate the promoter's activity in a cell-free system. The second aim will be to mutationally map the regulatory sequences in the cp35 promoter by deletion, linker scanning and site- directed mutagenesis. In order to identify the nuclear proteins involved in the observed transcriptional effects, cDNA cloning will be done for the third aim. Proteins will be identified by oligonucleotide screening of an expression library and/or affinity purification and peptide sequencing. The fourth and fifth aims of the project will be to address the hormonal regulation of the transcription factors cloned in aim 3. First we will measure the levels of mRNA for the factors(s) by Northern and in situ hybridization. Second we will produce antibodies to either transcription factor peptides or recombinant fusion proteins and then assess protein levels for the factor(s) by Western blotting. These experiments are to be of critical importance to understanding the transcriptional regulation of gene expression by polypeptide hormones. We can predict with a high degree of confidence that we will discover previously unidentified transcription regulatory factor(s) in these experiments. This confidence is based upon a substantial amount of preliminary data. The pigeon gene model is exceptionally productive for these experiments because of the specificity with which prolactin causes differentiative changes in gene expression and because of the accessibility of the system for biochemical and molecular biological study.